Monoclonal Antibody Self-Association, Cluster Formation, and Rheology at High Concentrations

Abstract

The rheological properties of macromolecular and colloidal suspensions are dependent on the thermodynamic and kinetic parameters that define viscous flow, and remain an active field of study with broad implications in cellular biophysics, soft-matter theory, and biopharmaceutical technology. Here we use static light scattering, small-angle X-ray scattering, and viscosity measurements as a function of protein concentration to semiquantitatively correlate the oligomeric state of an IgG1 antibody (mAb1) with its rheological behavior at solution pH 6.0 and varying ionic strength (modified by 0.01-0.1 M Na2SO4). Solution SAXS characterization of 100 mM Na2SO4 solutions confirmed that mAb1 forms reversible dimers with extended structures in dilute solutions. Light-scattering measurements over a wide range of concentrations (1-175 mg/mL) provide detailed information on the equilibrium thermodynamic mAb1 interactions and their modulation by modest increases of Na2SO4. Through the use of interacting hard sphere models to fit light-scattering data, we establish that protein cluster formations consisting of 2-9 mAb1 molecules also increase the viscosity of 175 mg/mL IgG solutions from 52 up to 450 cP. The analysis of dilute and semidilute mAb1 solution rheology correlates linearly with the thermodynamic equilibrium cluster size, consistent with the viscosity behavior of elongated oligomeric structures that are not significantly dendrimeric or in a state of globular collapse. Furthermore, SAXS- and rheology-based structural modeling illustrate that only a small set of anisotropic interactions between complementary surfaces are required to nucleate and propagate protein clusters.

Hydrogen exchange mass spectrometry reveals protein interfaces and distant dynamic coupling effects during the reversible self-association of an IgG1 monoclonal antibody

“…10 For example, Pathak et al demonstrated that the presence of reversibly associated clusters at high protein concentrations contributed to an increase in solution viscosity. 52 Similar trends in viscosity in response to changes in solution conditions that we describe here for mAb-C have been reported for other IgG1 mAbs, where the extent of viscosity increased in a concentration-dependent manner with increasing ionic strength 11,18,30,53,54 and solution pH, 11,55 related to elevated levels of protein RSA due to charge shielding effects. [56][57][58] The isoelectric point (pI) range of mAb-C is basic (pI»9.1-9.4), 45 and therefore, the overall surface charge of mAb-C is expected to be positive at neutral pH.…”

“…can either increase or decrease the extent of RSA of mAbs, depending on the distinct nature of the non-covalent interactions for an individual mAb formulated under specific conditions. 18,19 A variety of analytical tools have been used to characterize the RSA of proteins, and related effects on solution properties, including dynamic light scattering (DLS), 21 composition-gradient multi-angle static light scattering, 20,21 isothermal titration calorimetry, 15 surface plasmon resonance, 22 proton magnetic relaxation dispersion, 23 nuclear magnetic resonance, 24 fluorescence resonance energy transfer, 25 mass spectrometry (MS), 26 self-interaction nanoparticle spectroscopy, 27 size-exclusion chromatography, 28 analytical ultracentrifugation, 29 small angle X-ray scattering, 30 and atomic force microscopy. 31 Most of these measurements provide reliable data only at low-to-moderate protein concentrations (»1-20 mg/mL).…”

Molecular basis of high viscosity in concentrated antibody solutions: Strategies for high concentration drug product development

“…In qualitative agreement with the findings of Binabaji and coworkers, Lilyestrom et al also noted that clusters consisting of as many as nine antibody molecules are present in an antibody solution at 175 mg per mL that has viscosity of approximately 450 cP. 41 Additionally, recent computer simulations also suggested that higher-order antibody networks can form in certain antibody solutions at concentrations above 100 mg per mL. 42 The higher order networks and structures within liquids can be probed by oscillatory rheometry experiments using a time variant stress.…”

Characterization of mAb dimers reveals predominant dimer forms common in therapeutic mAbs

“…An elongated dimer was assigned a probable solution in SAXS measurements of dilute mAb solutions. 30 Furthermore, molecular dynamics simulation detected an elongated dimer as lowest energy minimum under certain conditions. 31 Elongated mAb dimers that are similar to those described here and were generated by process and light stress have been analyzed previously using TEM analysis.…”